CN113349196A - Mammary gland stem cell cryopreservation liquid and preparation method and cryopreservation method thereof - Google Patents
Mammary gland stem cell cryopreservation liquid and preparation method and cryopreservation method thereof Download PDFInfo
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- CN113349196A CN113349196A CN202110527330.6A CN202110527330A CN113349196A CN 113349196 A CN113349196 A CN 113349196A CN 202110527330 A CN202110527330 A CN 202110527330A CN 113349196 A CN113349196 A CN 113349196A
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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- A01N1/02—Preservation of living parts
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- A01N1/021—Preservation or perfusion media, liquids, solids or gases used in the preservation of cells, tissue, organs or bodily fluids
- A01N1/0221—Freeze-process protecting agents, i.e. substances protecting cells from effects of the physical process, e.g. cryoprotectants, osmolarity regulators like oncotic agents
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Abstract
The invention discloses a breast stem cell cryopreservation solution which comprises a solution A and a solution B, wherein the volume ratio of the solution A to the solution B is (1-3): 1; the solution A comprises the following components in parts by volume: 20-40 parts of glycerol and 60-80 parts of calf serum; the liquid B comprises the following components in parts by volume: 75-80 parts of DMEM/F12 basal medium, 10-20 parts of calf serum, 2 parts of vitamin A-free B-27, 0.5 part of heparin sodium, 0.4 part of insulin, 0.04 part of recombinant basic fibroblast growth factor, 0.05 part of epidermal cell growth factor and 0.01 part of Y-27632. The freezing storage liquid can obviously improve the freezing storage efficiency, reduce the ice crystal damage during the freezing storage of the cells, thereby improving the survival rate of the revived stem cells, maintaining the original dryness of the stem cells and keeping the multidirectional differentiation and self-renewal capacity of the stem cells.
Description
Technical Field
The invention belongs to the technical field of stem cell cryopreservation, relates to a mammary gland stem cell cryopreservation solution, also relates to a preparation method of the cryopreservation solution, and also relates to a cryopreservation method of the cryopreservation solution.
Background
The mammary gland stem cell is an adult stem cell with the capacity of multidirectional differentiation and self-renewal, and plays a crucial role in maintaining tissue homeostasis and generating breast cancer. In laboratory research and clinical application, the main preservation mode of the mammary gland stem cells is to mix the mammary gland stem cells with a cryoprotectant and then place the mixture in liquid nitrogen for low-temperature preservation. The most commonly used cryoprotectant in cell cryopreservation is dimethyl sulfoxide (DMSO), and too high concentration of DMSO has cytotoxic effect, and too low concentration may result in poor cryopreservation effect, thereby causing injury, loss of function and even death of breast stem cells. In addition, the frozen stock solution of stem cells needs to maintain the stem cell dryness as much as possible, and maintain the original multipotential differentiation and self-renewal ability, as compared with ordinary cells. Therefore, there is a need to develop a safe and reliable cryopreservation solution for mammary gland stem cells, which can solve the above-mentioned technical problems.
Disclosure of Invention
The invention aims to provide a cryopreservation solution for breast stem cells, which can be used for long-term cryopreservation of the breast stem cells, can be specifically applied to establishment and long-term preservation of a breast stem cell bank, and enables the recovered breast stem cells to keep relatively complete differentiation potential and higher survival rate.
The invention also provides a preparation method of the breast stem cell frozen stock solution;
the invention also provides a cryopreservation method of the breast stem cell cryopreservation liquid.
The first technical scheme adopted by the invention is that the breast stem cell cryopreservation solution comprises a solution A and a solution B, wherein the volume ratio of the solution A to the solution B is (1-3): 1; the liquid A comprises the following components in parts by volume: 20-40 parts of glycerol and 60-80 parts of calf serum;
the liquid B comprises the following components in parts by volume: 75-80 parts of DMEM/F12 basal medium, 10-20 parts of calf serum, 2 parts of vitamin A-free B-27, 0.5 part of heparin sodium, 0.4 part of insulin, 0.04 part of recombinant basic fibroblast growth factor, 0.05 part of epidermal cell growth factor and 0.01 part of Y-27632.
The first technical scheme of the invention is also characterized in that:
the volume ratio of the solution A to the solution B is 2-3: 1;
the solution A comprises the following components in parts by volume: 20-30 parts of glycerol and 70-80 parts of calf serum; the liquid B comprises the following components in parts by volume: 75 parts of DMEM/F12 basic culture medium, 20 parts of calf serum, 2 parts of vitamin A-free B-27, 2.5 parts of heparin sodium, 0.4 part of insulin, 0.04 part of recombinant basic fibroblast growth factor, 0.05 part of epidermal growth factor and 0.01 part of Y-27632.
The volume ratio of the solution A to the solution B is 2: 1; the liquid A comprises the following components in parts by volume: 25 parts glycerol and 75 parts calf serum;
the liquid B comprises the following components in parts by volume: 75 parts of DMEM/F12 basic culture medium, 20 parts of calf serum, 2 parts of vitamin A-free B-27, 2.5 parts of heparin sodium, 0.4 part of insulin, 0.04 part of recombinant basic fibroblast growth factor, 0.05 part of epidermal growth factor and 0.01 part of Y-27632.
The second technical scheme adopted by the invention is that the preparation method of the breast stem cell frozen stock solution specifically comprises the following steps:
step 1, dropwise adding glycerol in the solution A into calf serum under aseptic condition, uniformly mixing to obtain solution A, subpackaging, and storing at-20 ℃ for later use;
and 2, preparing a solution B under an aseptic condition, and storing the solution B at 4 ℃ for later use.
The third technical scheme adopted by the invention is that the cryopreservation method of the breast stem cell cryopreservation solution specifically comprises the following steps:
recovering the solution A at 37 ℃;
and step B, mixing the solution A and the solution B according to the ratio of 2: 1 to prepare a mixed solution;
step C, placing the breast stem cells to be cryopreserved into the mixed solution obtained in the step 2, and uniformly mixing to reach 1 x 106～1*109Final concentration in/mL.
The final concentration of the cryopreserved mammary stem cells in step C was 5 x 108/mL。
The frozen breast stem cells are P0-P9 generation stem cells.
The invention has the advantages that the cryopreservation liquid for the breast stem cells can greatly improve the cryopreservation efficiency, the average freeze-live rate reaches more than 90 percent, the ice crystal damage during cell cryopreservation can be obviously reduced, so that the live rate of the recovered stem cells is improved, the original dryness of the stem cells can be maintained, and the multidirectional differentiation and self-renewal capacity of the stem cells can be maintained. Meanwhile, the toxic effect of DMSO on cells is avoided, and the safety and stability in the cryopreservation process are improved.
Drawings
FIG. 1 is a 100-fold enlarged view of a revived stem cell in a cryopreservation solution for breast stem cells according to the present invention; FIG. 1a) is a 100-fold enlarged view of stem cells of comparative example 1; FIG. 1b) is a 100-fold magnification of stem cells of comparative example 2; FIG. 1c) is a 100-fold magnification of the stem cells of example 1; FIG. 1d) is a 100-fold magnification of the stem cells of example 2; FIG. 1e) is a 100-fold magnification of the stem cells of example 3; FIG. 1f) is a 100-fold magnification of the stem cells of example 4; FIG. 1g) 100-fold magnification of stem cells of example 5; FIG. 1h) 100-fold magnification of stem cells of example 6;
FIG. 2 is a 40-fold enlarged view of microspheres formed by stem cells in a breast stem cell cryopreservation solution according to the present invention; FIG. 2a) is a 40-fold magnification of microspheres formed in comparative example 1; FIG. 2b) is a 40-fold magnification of the microspheres formed in comparative example 2; FIG. 2c) is a 40-fold magnification of microspheres formed in comparative example 2; FIG. 2d) is a 40-fold magnification of microspheres formed in comparative example 2; FIG. 2e) is a 40-fold magnification of microspheres formed in comparative example 3; FIG. 2f) is a 40-fold magnification of microspheres formed in comparative example 4; FIG. 2g) is a 40-fold magnification of microspheres formed in comparative example 5; FIG. 2h) is a 40-fold magnification of microspheres formed in comparative example 6;
FIG. 3 is a clone diagram showing stem cell formation in comparative example 1 of a frozen stock solution of breast stem cells of the present invention;
FIG. 4 is a clone diagram showing stem cell formation in comparative example 2 of a frozen stock solution of breast stem cells of the present invention;
FIG. 5 is a clone diagram of the stem cell formation in example 1 of a frozen stock of mammary gland stem cells of the present invention;
FIG. 6 is a clone diagram of the stem cell formation in example 2 of a frozen stock of mammary gland stem cells of the present invention;
FIG. 7 is a clone diagram of the stem cell formation in example 3 of a frozen stock of mammary gland stem cells of the present invention;
FIG. 8 is a clone diagram of the stem cell formation in example 4 of a frozen stock of mammary gland stem cells of the present invention;
FIG. 9 is a clone diagram of the stem cell formation in example 5 of a frozen stock of mammary gland stem cells of the present invention;
FIG. 10 is a clone diagram of the stem cell formation in example 6 of the frozen stock solution of breast stem cells of the present invention.
Detailed Description
The present invention will be described in detail below with reference to the accompanying drawings and specific embodiments.
Example 1
A frozen stock solution of mammary gland stem cells is prepared by mixing solution A and solution B:
the volume ratio of the solution A to the solution B is 1: 1;
the solution A comprises the following components in parts by volume: 25 parts glycerol and 75 parts calf serum;
the liquid B comprises the following components in parts by volume: 75 parts DMEM/F12 basal medium, 20 parts calf serum, 2 parts vitamin A free B-27(50X), 2.5 parts heparin sodium (2. mu.g/mL), 0.4 part insulin (10. mu.g/mL), 0.04 part recombinant basic fibroblast growth factor (10ng/mL), 0.05 part epidermal growth factor (4ng/mL) and 0.01 part Y-27632(100 mM).
Example 2
The invention provides a breast stem cell cryopreservation solution, which is prepared by mixing solution A and solution B:
the volume ratio of the solution A to the solution B is 2: 1;
the solution A comprises the following components in parts by volume: 40 parts of glycerol and 60 parts of calf serum;
the liquid B comprises the following components in parts by volume: 75 parts DMEM/F12 basal medium, 20 parts calf serum, 2 parts vitamin A free B-27(50X), 2.5 parts heparin sodium (2. mu.g/mL), 0.4 part insulin (10. mu.g/mL), 0.04 part recombinant basic fibroblast growth factor (10ng/mL), 0.05 part epidermal growth factor (4ng/mL) and 0.01 part Y-27632(100 mM).
Example 3
The invention provides a breast stem cell cryopreservation solution, which is prepared by mixing solution A and solution B:
the volume ratio of the solution A to the solution B is 2: 1;
the solution A comprises the following components in parts by volume: 20 parts of glycerol and 80 parts of calf serum;
the liquid B comprises the following components in parts by volume: 75 parts DMEM/F12 basal medium, 20 parts calf serum, 2 parts vitamin A free B-27(50X), 2.5 parts heparin sodium (2. mu.g/mL), 0.4 part insulin (10. mu.g/mL), 0.04 part recombinant basic fibroblast growth factor (10ng/mL), 0.05 part epidermal growth factor (4ng/mL) and 0.01 part Y-27632(100 mM).
Example 4
The invention provides a breast stem cell cryopreservation solution, which is prepared by mixing solution A and solution B:
the volume ratio of the solution A to the solution B is 2: 1;
the solution A comprises the following components in parts by volume: 25 parts glycerol and 75 parts calf serum;
the liquid B comprises the following components in parts by volume: 80 parts DMEM/F12 basal medium, 15 parts calf serum, 2 parts vitamin A free B-27(50X), 2.5 parts heparin sodium (2. mu.g/mL), 0.4 parts insulin (10. mu.g/mL), 0.04 parts recombinant basic fibroblast growth factor (10ng/mL), 0.05 parts epidermal growth factor (4ng/mL) and 0.01 parts Y-27632(100 mM).
Example 5
The invention provides a breast stem cell cryopreservation solution, which is prepared by mixing solution A and solution B:
the volume ratio of the solution A to the solution B is 2: 1;
the solution A comprises the following components in parts by volume: 25 parts glycerol and 75 parts calf serum;
the liquid B comprises the following components in parts by volume: 75 parts DMEM/F12 basal medium, 20 parts calf serum, 2 parts vitamin A free B-27(50X), 2.5 parts heparin sodium (2. mu.g/mL), 0.4 part insulin (10. mu.g/mL), 0.04 part recombinant basic fibroblast growth factor (10ng/mL), 0.05 part epidermal growth factor (4ng/mL) and 0.01 part Y-27632(100 mM).
Example 6
A frozen stock solution of mammary gland stem cells is prepared by mixing solution A and solution B:
the volume ratio of the solution A to the solution B is 3: 1;
the solution A comprises the following components in parts by volume: 25 parts glycerol and 75 parts calf serum;
the liquid B comprises the following components in parts by volume: 75 parts DMEM/F12 basal medium, 20 parts calf serum, 2 parts vitamin A free B-27(50X), 2.5 parts heparin sodium (2. mu.g/mL), 0.4 part insulin (10. mu.g/mL), 0.04 part recombinant basic fibroblast growth factor (10ng/mL), 0.05 part epidermal growth factor (4ng/mL) and 0.01 part Y-27632(100 mM).
The preparation method of the stem cell cryopreservation solution of the embodiment 1-6 mainly comprises the following steps:
(1) slowly adding glycerol in solution A into calf serum under aseptic condition, mixing well to obtain solution A, subpackaging, and storing at-20 deg.C for use;
(2) preparing solution B under aseptic condition and storing at 4 deg.C for use
The cryopreservation method of the breast stem cell cryopreservation solution provided by the preferred embodiment of the invention comprises the following steps:
(1) recovering solution A at 37 deg.C;
(2) mixing the solution A and the solution B according to the ratio of 3: 1 to prepare a mixed solution;
(3) mixing the breast stem cells to be frozen in the mixed solution to 5 x 108Final concentration of/mL, 1mL per tube, 4 replicates per group, cryopreserved mammary stem cells as primary stem cells.
Comparative example 1
A conventional cell freezing medium is composed of calf serum and dimethyl sulfoxide, and the volume ratio of the calf serum to the dimethyl sulfoxide is 9: 1.
comparative example 2
A frozen stock solution of mammary gland stem cells is prepared by mixing solution A and solution B:
the volume ratio of the solution A to the solution B is 3: 1;
the solution A comprises the following components in parts by volume: 25 parts glycerol and 75 parts calf serum;
the liquid B is DMEM/F12 basic culture medium.
Experiment of
And (3) respectively freezing the primary breast stem cells obtained by sorting through a flow cytometer by using the freezing medium of the comparison example of the embodiment of the invention, putting the freezing tube into a programmed cooling box which is restored to room temperature, putting the freezing tube into a refrigerator at minus 80 ℃ for overnight, and transferring the primary breast stem cells into a liquid nitrogen tank the next day. Taking out and recovering after being frozen for 1 month, counting the number and proportion of the recovered living cells by trypan blue staining, plating the cells in a low-adsorption 96-well plate required by microspheres formed by stem cells at the density of 2000 cells/hole to culture and feed layer cells, analyzing the dryness maintenance level of the recovered stem cells by counting the microspheres and clones formed by the stem cells, and setting 4 parallel cells in each group. After recovery, the 100-fold enlarged view of stem cells is shown in fig. 1(a) - (h), the 40-fold enlarged view of microspheres formed by stem cells is shown in fig. 2(a) - (h), the clone formed by stem cells in comparative example 1 is shown in fig. 3, the clone formed by stem cells in comparative example 2 is shown in fig. 4, the clone formed by stem cells in example 1 is shown in fig. 5, the clone formed by stem cells in example 2 is shown in fig. 6, the clone formed by stem cells in example 3 is shown in fig. 7, the clone formed by stem cells in example 4 is shown in fig. 8, the clone formed by stem cells in example 5 is shown in fig. 9, and the clone formed by stem cells in example 6 is shown in fig. 10.
The results of measuring the viability of the stem cells and the number and diameter of the microspheres formed by the stem cells are shown in Table 1.
TABLE 1 recovery rates of cells at different time points
As can be seen from table 1, there is no significant difference between control 1 and control 2, indicating that there is no significant difference between the two conventional cryopreservation methods. Compared with the comparative example, in examples 1-6, after freezing recovery in examples 5 and 6, the freezing efficiency of the freezing medium on cells is significantly higher than that of the comparative example, wherein the freezing efficiency of the freezing medium in example 6 is the highest; after the freezing recovery of the examples 2, 3 and 4, the freezing efficiency of the freezing solution to the cells is not obviously different from that of the control example. The stem cell cryopreservation liquid formula of the invention is superior to the cell cryopreservation effect of the conventional cell cryopreservation liquid. In addition, the microsphere number and the microsphere diameter of the mammary gland stem cells obtained by the stem cell cryopreservation liquid formulas of the examples 1 to 6 are obviously higher than those of the control example, which proves that the stem cell cryopreservation liquid formulas of the examples have better dryness maintaining effect on the mammary gland stem cells. And after the examples 2, 3, 4 and 5 are subjected to cryopreservation and recovery, the microsphere number and the microsphere diameter formed by the stem cells are obviously higher than those of the comparative examples 1 and 2 and lower than those of the example 6. This shows that the ratio of each component in the stem cell cryopreservation solution can also significantly affect the cryopreservation effect of the stem cell cryopreservation solution on the stem cells.
FIG. 1(a) - (h) is a 100-fold enlarged view of stem cells after recovery, and it can be seen from the images that the cryopreservation efficiency of the cryopreservation solution of the examples 1-6 is significantly higher than that of the control example compared with the control example, wherein the cryopreservation efficiency of the cryopreservation solution of the example 6 is the highest.
Fig. 2(a) - (h) are 40-fold enlarged views of microspheres formed by stem cells after recovery, and it can be seen from the drawings that the number of microspheres and the diameter of microspheres of the breast stem cells obtained by the stem cell cryopreservation liquid formulations in examples 1-6 are significantly higher than those of the control example, wherein the dry maintenance effect of the stem cell cryopreservation liquid formulation in example 6 on the breast stem cells is better.
Fig. 3 to 10 are clone graphs formed by the stem cells after recovery, and it can be seen from the graphs that the clone number of the breast stem cells obtained by the stem cell cryopreservation liquid formula of the embodiments 1 to 6 is significantly higher than that of the comparative example, wherein the dryness maintenance effect of the stem cell cryopreservation liquid formula of the embodiment 6 on the breast stem cells is better.
Claims (7)
1. A breast stem cell cryopreservation liquid is characterized in that: the liquid A and the liquid B are included, and the volume ratio of the liquid A to the liquid B is 1-3: 1; the liquid A comprises the following components in parts by volume: 20-40 parts of glycerol and 60-80 parts of calf serum;
the liquid B comprises the following components in parts by volume: 75-80 parts of DMEM/F12 basal medium, 10-20 parts of calf serum, 2 parts of vitamin A-free B-27, 0.5 part of heparin sodium, 0.4 part of insulin, 0.04 part of recombinant basic fibroblast growth factor, 0.05 part of epidermal cell growth factor and 0.01 part of Y-27632.
2. The cryopreservation solution for the breast stem cells according to claim 1, wherein: the volume ratio of the liquid A to the liquid B is 2-3: 1;
the liquid A comprises the following components in parts by volume: 20-30 parts of glycerol and 70-80 parts of calf serum; the liquid B comprises the following components in parts by volume: 75 parts of DMEM/F12 basic culture medium, 20 parts of calf serum, 2 parts of vitamin A-free B-27, 2.5 parts of heparin sodium, 0.4 part of insulin, 0.04 part of recombinant basic fibroblast growth factor, 0.05 part of epidermal growth factor and 0.01 part of Y-27632.
3. The cryopreservation solution for the breast stem cells according to claim 2, wherein: the volume ratio of the solution A to the solution B is 2: 1; the liquid A comprises the following components in parts by volume: 25 parts glycerol and 75 parts calf serum;
the liquid B comprises the following components in parts by volume: 75 parts of DMEM/F12 basic culture medium, 20 parts of calf serum, 2 parts of vitamin A-free B-27, 2.5 parts of heparin sodium, 0.4 part of insulin, 0.04 part of recombinant basic fibroblast growth factor, 0.05 part of epidermal growth factor and 0.01 part of Y-27632.
4. The method for preparing the cryopreservation solution of the breast stem cells according to any one of claims 1 to 3, which comprises the following steps:
step 1, dropwise adding glycerol in the solution A into calf serum under aseptic condition, uniformly mixing to obtain solution A, subpackaging, and storing at-20 ℃ for later use;
and 2, preparing a solution B under an aseptic condition, and storing the solution B at 4 ℃ for later use.
5. The cryopreservation method of breast stem cell cryopreservation liquid according to any one of claims 1 to 3, comprising the following steps:
recovering the solution A at 37 ℃;
and step B, mixing the solution A and the solution B according to the ratio of 2: 1 to prepare a mixed solution;
step C, placing the breast stem cells to be cryopreserved into the mixed solution obtained in the step 2, and uniformly mixing to reach 1 x 106～1*109Final concentration in/mL.
6. The cryopreservation method of the breast stem cell cryopreservation liquid according to claim 5, which comprises the following steps: the final concentration of the cryopreserved mammary gland stem cells in the step C is 5 x 108/mL。
7. The method of claim 6, wherein the cryopreserved breast stem cells are P0-P9 generation stem cells.
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| GB734368A (en) * | 1953-01-29 | 1955-07-27 | Medical Res Council | Improvements in or relating to the preservation of blood |
| CN107494517A (en) * | 2017-08-07 | 2017-12-22 | 章毅 | Serum-free frozen stock solution and its application in mescenchymal stem cell is frozen |
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Application publication date: 20210907 |